Vertebrates have developed a sophisticated system to protect themselves against a wide variety of hazards including various viruses and microorganisms, such as bacteria and fungi, as well as genetic diseases, neoplasia, and effects of a variety of toxins. The system has evolved based on the ability to recognize self as distinct from non-self or “foreign.” A broad panoply of defense mechanisms are involved, including phagocytosis, lysis, such as complement mediated or perform mediated lysis, and killer cells, such as cytotoxic T-lymphocytes (CTLs; also known as cytotoxic/suppressor T-cells, Tc/s), natural killer cells, antibody dependent cytotoxic cells, and the like. Various cell types offer different mechanisms whereby the invader or endogenous diseased cell may be eliminated.
A key to the immune defensive mechanism is the T-cell. For instance, it is well known that the adaptive immune system shows a much stronger response on second, as compared to first, encounter with an antigen. This phenomenon is exploited in vaccination, which works by inducing a state of lasting immunity known as immunological memory. Immunological memory requires activation of T-lymphocytes specific for the vaccine antigen.
T-cells have been found to be “restricted” in that they respond to an antigen in relation to one or a few specific molecules (now called major histocompatibility or MHC molecules) associated with their natural host. In vitro, T-cells from a host of one haplotype respond to an antigen in relation to an MHC molecule of a different haplotype host. The T-cell receptor recognition repertoire appears to be narrower than the recognition repertoire of immunoglobulins produced by B-cells. In addition, rather than directly binding to an antigen as do antibodies and other immunoglobulins, the T-cell receptor appears to require concomitant binding to a foreign antigen and an MHC molecule.
MHC molecules are divided into two classes, Class I and Class II. The former class is relatively ubiquitous on vertebrate cells, while the latter is generally limited to lymphocytes, macrophages, and dendritic cells. Functionally different T-cells appear to be activated in relation to one or the other class of MHC molecules. The nature of the activity of a T-cell varies with the Class of the MHC molecule to which it is complementary. A T-cell clone recognizes a specific antigen in conjunction with a specific MHC allele. Furthermore, variation in the antigen structure affects the nature of the response when the T-cell, antigen, and antigen presenting cell are brought together. Depending upon the nature of the structural change, three possibilities are encountered: no change, increased stimulation or decreased stimulation of an immune response to the antigen.
T-lymphocytes detect foreign polypeptide antigens by recognizing—via the T-cell receptor (“TCR”)—peptide fragments derived from the antigen. Most T-lymphocytes, however, are MHC restricted, that is, they recognize only complexes of peptides bound to the highly polymorphic membrane proteins encoded by Class I and Class II MHC genes and presented (displayed) on the surface of an accessory cell (designated an antigen-presenting cell or “APC”), in which the antigen has been processed.
Antigens can be processed by one of two pathways, depending on their origin, inside or outside the APC. In a first pathway, foreign material from outside a cell is engulfed by a specialized APC (often a macrophage or B-cell), which breaks down the material and complexes the processed antigen with Class II MHC molecules. In particular, MHC Class II molecules are synthesized in the endoplasmic reticulum with their antigenic peptide binding sites blocked by the invariant chain protein (Ii). These MHC Class II-Ii protein complexes are transported from the endoplasmic reticulum to a post-Golgi compartment where Ii is released by proteolysis and a specific antigenic peptide becomes bound to the MHC Class II molecule.
Class II MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T-lymphocytes, B-lymphocytes, and macrophages. Complexes of Class II MHC molecules and immunogenic peptides are recognized by helper T-lymphocytes (also known as helper/accessory T-cells, “Th”) and induce proliferation of Th lymphocytes. Class II MHC complexes also stimulate secretion of cytokines by Th cells, resulting in amplification of the immune response to the particular immunogenic peptide that is displayed. Th1 cells produce interferon-γ and other cytokines that stimulate CTLs, while other cytokines produced by Th2 cells help B-cells to produce antibodies.
A second antigen processing pathway is generally involved with foreign or aberrant proteins made within cells, such as virus-infected or malignant cells. These proteins are subjected to partial proteolysis by the proteosome within such cells, so as to form peptide fragments that then associate with Class I MHC molecules and are transported to the cell surface for presentation to T-cells. Class I MHC molecules are expressed on almost all nucleated cells, and complexes of Class I MHC molecules and bound immunogenic peptides are recognized by CTLs, which then destroy the antigen-bearing cells. CTLs are particularly important in tumor rejection and in fighting viral infections.
For a CTL to recognize an antigen in the form of a peptide fragment bound to the MHC class I molecule, that antigen must normally be endogenously synthesized by the cell and a portion degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with Class I heavy chains to facilitate proper folding and association with the subunit 132 microglobulin. The peptide-MHC Class I complex is then routed to the cell surface for expression and potential recognition by specific CTLs.
By these dual antigen processing pathways, appropriate defenses are generated against both exogenous and internally produced antigens. Thus, antigens taken up from the extracellular environment eventually elicit B-cells to produce antibodies that protect the organism against a subsequent challenge by an agent comprising the exogenous antigen. On the other hand, antigens comprised of abnormal structures made within an abnormal or errant cell (for example a virus-infected or malignant cell) activate an immune response that eventually leads to killing of the errant cell. There is considerable interest in methods for better stimulating immune responses to antigens that are processed by either of these two pathways and presented by either MHC Class I or Class II molecules.
In view of the above knowledge, it is understandable that there has been substantial interest in using short peptides to affect an immune response in vivo and in vitro, to provide stimulation or inactivation of a particular response. Thus, appropriate immunogenic peptides might modulate a natural immune response to a particular event, either by activating particular lymphocytes to enhance a protective response or by deactivating particular lymphocytes to diminish or prevent an undesirable response.
The human immunodeficiency virus (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders (see, for example, Barre-Sinoussi et al., Science 220:868-871, 1983; Gallo et al., Science 224:500-503, 1984; Levy et al., Science 225:840-842, 1984; Siegal et al., N. Engl. J. Med 305:1439-1444, 1981). AIDS patients usually have a long asymptomatic period followed by the progressive degeneration of the immune system and the central nervous system. Replication of the virus is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture (Zagury et al., Science 231:850-853, 1986). Molecular studies of HIV-1 show that it encodes a number of genes (Ratner et al., Nature 313:277-284, 1985; Sanchez-Pescador et al., Science 227:484-492, 1985), including three structural genes—gag, pol and env—that are common to all retroviruses. Nucleotide sequences from viral genomes of other retroviruses, particularly HIV-2 and simian immunodeficiency viruses (SIV; previously referred to as STLV-III), also contain these structural genes (Guyader et al., Nature 326:662-669, 1987; Chakrabarti et al., Nature 328:543-547, 1987).
Development of an effective HIV vaccine is a major challenge due to antigenic variation and immune escape mechanisms. Strategies that include the use of recombinant DNA technology and novel antigen delivery methods are being applied to the development of HIV vaccines. Most HIV-1 vaccine constructs (DNA and recombinant protein vaccine) are subtype-specific and designed to prime only one arm of the immune system, that is, CTL responses or humoral B-cell responses. Emerging data suggest that broadly reactive T-cell responses, as well as neutralizing antibody responses are likely to be required for an effective immune response against HIV-1. Additionally, current human phase III vaccine trials using recombinant envelope proteins, suggest that immunity to HIV-1 envelope proteins is probably not sufficient for complete protection against HIV-1. Thus the results from multiple studies suggest that additional epitopes as well as activation of both arms of the immune system may be required for an effective HIV-1 vaccine.
By way of one example of peptide immunogens, Peter et al. (Vaccine 19:4121-4129, 2001) disclose induction of a CTL response against multiple CTL epitopes present in HIV proteins using short synthetic peptides. Four IHLA-A2.1 restricted peptides (RT 476-484, p17 77-85, gp41 814-823, RT 956-964) that showed stable binding to the HLA-A2.1 molecule in an in vitro binding assay were able to elicit a strong specific immune response in HLA-A2.1 transgenic mice when injected with a peptide (“P30”) used as a universal T-cell helper epitope, in incomplete Freund adjuvant (IFA) or a nonionic emulsifier (Montanide™ ISA 720). The use of biodegradable poly-L-glutamic acid (PLGA) microspheres (MS) as adjuvant was also successfully tested for all peptides.
Many studies of cross-clade recognition of HIV epitopes have been carried out (see, for example, Wilson et al., AIDS Res. Hum. Retroviruses 14:925-937, 1998; McAdam et al., AIDS 12:571-579, 1998; Lynch et al., J Infect Dis. 178:1040-1046, 1998; Boyer et al. Dev. Biol. Stand. 95:147-53, 1998; Cao et al., J. Virol. 71:8615-8623, 1997; Durali et al., Viral. 72:3547 3553, 1998). These studies often used whole-gene, vaccinia-expressed constructs to probe CTL lines from HIV-1 infected or HIV-1 vaccinated volunteers for CTL responses. What appeared to be cross-clade recognition by CTLs in these experiments may have been recognition of CTL epitopes that are conserved within the large gene constructs cloned into the vaccinia constructs and into the vaccine strain (or the autologous strain). Where responses to specific peptides, and their altered sequences in other HIV strains, have been tested, and the peptides have been mapped, some studies have shown a lack of cross-strain recognition (Dorrel et al., HIV Vaccine Development Opportunities And Challenges Meeting, Abstract 109 (Keystone, Colo., January 1999)). Studies of virus escape from CTL recognition carried out on HIV-1 infected individuals have also shown that viral variation at the amino acid level may abrogate effective CTL responses (Koup, J. Exp. Med. 180:779-782, 1994; Dai et al., J. Virol. 66:3151-3154, 1992; Johnson et al., J. Exp. Med. 175:961-971, 1992).
Unfortunately, existing candidate HIV-1 vaccines are subtype specific, and are expected not protect against diverse natural HV-1 infections. This is true of both DNA vaccine constructs as well as recombinant protein vaccines. Furthermore, many of the existing constructs have focused on priming only one arm of the immune system, that is, cell mediated T-cell responses or humoral B-cell responses. In addition, while some DNA constructs have shown promising results in lowering viremia in animal model systems, none has been able to confer sterilizing immunity. These data suggest that both B-cell and T-cell responses may be needed for a protective immune response against HIV-1. Additionally, current human phase 3 vaccine trials using recombinant envelope proteins, suggests that immunity to HIV-1 envelope proteins is probably not sufficient for complete protection against HIV-1. Prime-boost strategy using recombinant envelope from HIV-1 subtype B also has not been successful in boosting the immune responses.
As the HIV epidemic continues to spread world wide, the need for effective immune-stimulatory compositions and vaccines remains urgent.